TOXICOLOGY AND APPLIED PHARMACOLOGY 108,78-85 (1991)

Neurotoxic Effects of Two Different Pyrethroids, Bioallethrin and Deltamethrin, on Immature and Adult Mice: Changes in Behavioral

and Muscarinic Receptor Variables

PER ERIKSSON* AND ANDERS FREDRlKssoNt

*Department of Zoophysiology, Uppsala University, Box 560, S-751 22 Uppsala. Sweden; and TDepartment of Toxicology. Uppsala University, Box 594. S-751 24 Uppsala, Sweden

Received June I I. 1990; accepted November 7. 1990

Neurotoxic Effects of Two Different Pyrethroids. Bioallethrin and Deltamethrin, on Immature and Adult Mice: Changes in Behavioral and Muscarinic Receptor Variables. ERIKSSON, P., AND

FREDRIKSON, A. (1991). Toxicol. Appl. Pharrnacol. 108, 78-85. We have recently shown that two pyrethroids, bioallethrin and deltamethrin. affect muscarinic cholinergic receptors (MAChR) in the neonatal mouse brain when given to suckling mice during the period of rapid brain growth. Such early exposure to these pyrethroids can also lead to permanent changes in the MAChR and behavior in the mice as adults. In the present study, male NMRI mice were given bioallethrin (0.7 mg). deltamethrin (0.7 mg), or a 20% fat emulsion vehicle (10 ml) per kilogram of body weight per OS once daily between the 10th and 16th postnatal day. The mice were subjected to behavioral tests upon reaching the age of I7 days and at 4 months. Within l-2 weeks after the behavioral tests the mice were killed by decapitation and crude synaptosomal fractions (P2) were prepared from the cerebral cortex, hippocampus. and striatum. The densities of MAChR were assayed by measuring the amounts of quinuclidinyl benzilate ([‘HIQNB) specifically bound in the P2 fraction. The proportions of high-affinity (HA) and low-affinity (LA) binding sites of MAChR were assayed in a displacement study using [‘H]QNB/carbachol. The behavioral tests at an adult age of 4 months indicated a significant increase in spontaneous motor behavior in both bioallethrin- and deltamethrin-treated mice. There was also a significant decrease and a tendency toward a decrease in the density of MAChR in the cerebral cortex in mice receiving bioallethrin and deltamethrin, respectively. The proportions of HA- and LA-binding sites of MAChR were not changed. This study further supports that disturbances ofthe cholinergic system during rapid development in the neonatal mouse can lead to permanent changes in cholinergic and behavioral variables in the animals as adults. o 1991 Academic PIW. ITIC.

Pyrethroid insecticides are neurotoxicants that is tremor. while choreoathetosis and salivation have achieved widespread agricultural and are the main symptoms of the latter. Obser- environmental health applications, due to vation of nonmammalian nerve membrane their strongly insecticidal properties. The py- preparations has revealed that one of the basic rethroids are derivatives of natural pyrethrins mechanisms of pyrethroid action involves in- (Elliott et al.. 1967; Elliott, 1977) and are terference with nerve membrane sodium commonly divided into Type I compounds (or channels, leading to prolonged depolarization T-syndrome pyrethroids) which lack an (Y-cy- and induction of repetitive firing (Type I), or ano substituent, and Type II compounds (or depolarization block resulting in nerve inex- CS-syndrome pyrethroids), which contain an citability (Type II) (Wouters and van den a-cyanophenoxybenzyl substituent (Gammon Bercken, 1978: Lund and Narahashi, 1983; et al., 198 1; Verschoyle and Ah-idge, 1980). Vijverberg and van den Bercken, 1982; Nar- The main symptom of exposure to the former ahashi, 1985). Effects on sodium channels

004 1-008X/9 1 $3.00 Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.

78

PYRETHROIDS: NEONATAL AND ADULT NEUROTOXICITY 79

have also been observed in the mouse and rat brain (Ghiasuddin and Soderlund, 1985; Brown et al., 1988) and in mammalian neu- roblastoma cells (Roche et al., 1985).

During the development of a mammal there are periods which may be critical for its normal maturation, e.g., the period of rapid brain growth-the “brain growth spurt” (Davison and Dobbing, 1968) when the maturation of axonal and dendritic outgrowths and the es- tablishment of neural connections, synapto- genesis, take place. This neonatal stage of de- velopment is also associated with a large num- ber of biochemical changes that will transform the feto-neonatal brain into that of the mature adult. The rodent cholinergic nerve cells em- ploy a transmitter system that undergoes rapid development during the brain growth spurt (Coyle and Yamamura, 1976; Falkebom et al., 1983; Marchi et al., 1983; Hohmann et al., 1985; Hijhmann and Ebner, 1985) and is therefore a system that can be susceptible to disturbances.

We have recently observed that two pyre- throids of different types, bioallethrin (Type I) and deltamethrin (Type II), affect the cholin- ergic system in the neonatal mouse brain (Er- iksson and Nordberg, 1990). At doses below those causing neurotoxic symptoms, both af- fected the muscarinic cholinergic receptors (MAChR) in the cerebral cortex. The effect of bioallethrin on the MAChR in the neonatal mouse brain was the same as those seen earlier in neonatal mice exposed to DDT (Eriksson and Nordberg, 1986). DDT and pyrethroids (Type I) show similarities with respect to both neurotoxic symptoms and neurotoxic action (Verschoyle and Alridge, 1980; Narahashi, 1982; Woolley, 1982; Casida et al., 1983). Concerning DDT we have recently reported that neonatal exposure to a single, low, oral dose of DDT leads to behavorial deviations, such as hyperactivity, in the adult mouse, changes in the cholinergic system, such as in- creased release of acetylcholine (ACh), and a tendency toward a decrease in the MAChR (Eriksson et al., 1990a,b).

The present study was undertaken to in- vestigate whether two pyrethroids, bioallethrin (Type I) and deltamethrin (Type II), will affect the MAChR in the adult mouse brain and modify the behavior of the young and adult animal when given to neonatal mice during the brain growth spurt.

METHODS

Ten-day-old NMRI mice of both sexes, which were off- spring of pregnant mice purchased from ALAB, Sweden, were used. Each litter, adjusted within 24 hr to 8- 10 mice by euthanizing excess pups, was kept together with its re- spective mother in a plastic cage in a room at a temperature of 22°C and a 12/12-hr light/dark cycle. At the age of 4 weeks the mice were weaned and the mate mice were placed and raised in groups of 4 in a room for only male mice. The animals were supplied with standardized pellet food (Ewos, Sweden) and tap water ad libitum.

Bioallethrin (2-allyl-4-hydroxy-3-methyl-2cyclopenten- 1 -one-( 1 R,3R,4S)-2,2-dimethylcyclopropanecarboxylate) (MW302)anddeltamethrin(S-u-cyano-3-phenoxybenzyl- (I R)-cis-3(2,2-dibromovinyl)-2,2-dimethylcyclopropane- carboxylate) (MW 505) were generously donated by Dr Tessier (Rouse11 Uclaf. France). The substances were dis- solved in a mixture of egg lecithin (Merck) and peanut oil (Oleum arachidis) (I : 10 w:w) and were thereafter sonicated together with water to yield a 20% (w:w) fat emulsion ve- hicle containing deltamethrin at the concentration of 0.07 mg/ml or bioallethrin at the concentration of 0.07 mp/ ml. The substances were administered orally via a PVC tube (diameter 1 .O mm) as one single dose per day for 7 days to both male and female mice as in our previous study (Eriksson and Nordberg, 1990). This was done in order to simulate the exposure time effective for DDT (Eriksson and Nordberg, 1986). The amounts of pyre- throids given were as follows; deltamethrin, 0.7 mg (1.4 pmol)/kg body wt; bioallethrin, 0.7 mg (2.4 rmol)/kg body wt. Mice serving as controls received 10 ml/kg body wt of the 20% fat emulsion vehicle in the same manner. Each treatment group consisted of mice from three to four dif- ferent litters (Denenberg, 1984).

Behavioral tesfing. Spontaneous behavior was tested in male mice at the age of 17 days and 4 months as previously described (Eriksson et al., 1990a,b). The animals were only tested once and the tests were performed between 08:00- 12:OO A.M. under the same light and temperature condi- tions as the housing. Motor activity was measured over 3 X 20 min in an automated device consisting of cages (40 X 25 X 15-cm) placed within two series of infrared beams (low level and high level) (Rat-0-Matic, ADEA Elektronik AB, Uppsala, Sweden) (Archer et al., 1987). Locomotion counting occurred when the mouse moved horizontally through the low-level grid of infrared beams. Rearing was

80 ERIKSSON AND FREDRIKSSON

registered as vertical movement at a rate of four counts per second, as long as a single high-level beam was inter- rupted, i.e., the number of counts obtained was propor- tional to time spent in rearing up. Activity was registered by a pick-up (mounted on a lever with a counterweight) with which the test cage was in contact. The pick-up reg- istered all types ofvibration within the test cage, i.e., those caused by mouse movements, shaking (tremors), and grooming. By means of this equipment. the spontaneous behavior of the 17-day-old mouse, only variable loco- motion was used.

Receptor assu~: The mice were killed by decapitation after the test for behavior changes at an adult age of about 4 months. A crude synaptosomal P2 fraction (Gray and Whittaker, 1962) from the cerebral cortex, hippocampus. and striatum with a protein content of about 2 mg/ml (measured according to Lowry e( al., 195 1) was prepared as earlier described (Eriksson and Nordberg, 1986). The P2 fractions were kept frozen (-25°C) until assayed (within 4 months).

Measurements of muscarinic receptor density and pro- portions of muscarinic high-affinity (HA) and low-affinity (LA) binding sites were performed following the methods of Nordberg and Winblad (198 1) and Nordberg and Wahlstrom (I 982) respectively, as earlier described by Eriksson and Nordberg (1986). Briefly. the assays were performed by measuring tritium-labeled quinuclidinyl benzilate ([3H]QNB; sp act 1.44 TBq/mmol, 0.2 and 0. I nM, respectively, Amersham International plc. UK) spe- cifically bound in the P2 fraction by using atropine ( 10m4 M) for measuring the nonspecific binding, and different concentrations of carbachol (IO-*- IO-* M) for measuring the proportions of high- and low-affinity binding sites, re- spectively. The specific binding constituted about 98% of the total [3H]QNB binding. To analyze the proportions of the high- and low-affinity binding sites. the data of the competitive displacement of [3H]QNB by carbachol were fitted by a nonlinear least-squares method as described by Birdsall et a/. (1978). The computerized model in Graph Pad was used to calculate the percentages of high- and low-affinity binding sites with corresponding affinity con- stants.

Statistical andysix Muscarinic receptors; Student’s I test was used in evaluating differences between controls and treated animals, except for the evaluation ofthe affinity constants, where the Mann-Whitney V test was used.

Behavioral data were analyzed using ANOVA with a split-plot design (Kirk, 1968). Pairwise testing between deltamethrin. bioallethrin, and control groups was per- formed with the Tukey HSD tests (Kirk. 1968).

RESULTS

No clinical signs of pyrethroid poisoning were observed in the treated mice throughout

the experimental period. Nor were there any significant deviations in weight gain [g, mean + SD, (n)] between the bioallethrin [35.95 + 3.46 (21)]- or deltamethrin [38.04 + 4.09, ( i7)]-treated mice, compared with the vehicle [38.40 * 4.14, (17)]-treated mice.

The results from locomotion, rearing, and total activity variables in 4-month-old NMRI male mice after repeated daily administration of bioallethrin and deltamethrin 0.7 mg/kg body wt per OS when they were 10 days old for 7 days, or controls receiving in the same manner 10 ml/kg body wt of the 20% fat emulsion vehicle are given in Fig. 1. The be- havioral tests revealed significant deviations by the bioallethrin- and deltamethrin-treated mice, compared with controls. There were sig- nificant group X period interactions [F(4,66) = 6.12, F(4,66) = 26.99, F(4,66) = 20.751 for the locomotion, rearing, and activity variables, respectively. Pairwise testing between bioal- lethrin, deltamethrin, and control groups was performed with the Tukey HSD tests and showed a significant (p G 0.0 1) difference dur- ing the last 20-min period in all three test vari- ables in bioallethrin-treated mice. In the del- tamethrin-treated mice a significant difference was observed in the variables locomotion and total activity. In the 17-day-old mice, in con- trast to the 4-month-old mice, there was no significant change in the behavioral variable locomotion between the pyrethroid-treated mice and corresponding controls.

About 1 week after the behavioral tests on the 4-month-old mice, the animals were killed and the MAChR were assayed in the cerebral cortex, hippocampus, and striatum. The den- sities of the MAChR are given in Table 1. There was a significant decrease (a G 0.05) and a tendency toward a decrease (0.05 < p < 0.1) in the amount of specific [3H]QNB- binding sites in the cerebral cortex in mice re- ceiving bioallethrin and deltamethrin, respec- tively. In the other two brain regions, hippo- campus and striatum, no significant changes were observed after bioallethrin or deltameth- r-in treatment.

PYRETHROIDS: NEONATAL AND ADULT NEUROTOXICITY 81

600 ‘s 500

5 400

; 300 l *

z 200

5 100 0

123 123 123

800 ] **

= iii

600

z 0 z

400

% 2 200

0 123 123 123

5000,

i 3000

5 2000

2 1000 P

0 123 123 123

O-20 20-40 40-60

TIME(MIN)

FIG. 1. Spontaneous behavior in 4-month-old NMRI male mice after repeated daily administration of either bioallethrin (0.7 mg). deltamethrin (0.7 mg), or the 20% fat emulsion vehicle (10 ml) per kilogram of body weight per OS at 10 days old for 7 days. Each treatment group contained I2 mice from three different litters. Measure- ment of motor activity, see Methods. Statistical analysis of behavioral data was performed using ANOVA with a split-plot design (Kirk, 1968). There were significant group X period interactions [F(4,66) = 6.12. F(4.66) = 26.99, F(4.66) = 20.751 for the locomotion. rearing, and activity variables, respectively. Pairwise testing between bioalleth- tin. deltamethrin. and control groups was performed with the Tukey HSD tests (Kirk, 1968). The treatment groups are indicated by: I, control; 2. deltamethrin; 3, bioallethrin and the statistical difference from the control is indicated by **P G 0.01.

The observation made of the specific [3H]QNB binding in the cerebral cortex was further explored in a competition experiment. The proportions of HA- and LA-binding sites and the affinity constants of the muscat-uric receptors were determined in the antagonist ([3H]QNB)/agonist (carbachol) competition assay by using different concentrations of car- bachol. The two-site model used in previous experiments (Eriksson and Nordberg, 1986, 1990) was used to evaluate the competition data, which are given in Table 2. No significant change was observed either in the proportion of HA- and LA-binding sites or the affinity constants in mice treated with bioallethrin or deltamethrin. compared with control.

DISCUSSION

The present investigation shows that neo- natal exposure to bioallethrin (0.7 mg/kg bw) or deltamethrin (0.7 mg/kg bw) between the 10th and 16th postnatal day produces signif- icant behavioral deviations in the adult mouse at the age of 4 months. The cholinergic system was also affected in the same brain region as earlier observed in the neonatal mouse, namely the cerebral cortex, in mice exposed to bioallethrin (Eriksson and Nordberg, 1990). Contrary to the change in the neonatal mouse a decrease in the amount of MAChR without any significant change in the proportions of HA- and LA-binding sites was found in the adult mouse. Even the minor differences in potency between bioallethrin and deltameth- tin, reflected in the behavior and MAChR in the adult mouse, were also seen in the MAChR in neonatal mouse brain (Eriksson and Nord- berg, 1990).

The behavioral data show a disruption in habituation, in adult mice exposed to bioal- lethrin or deltamethrin as neonates. Thus, ha- bituation, defined by a decrease in locomotion, rearing, and total activity variables in response to the diminished novelty of the test chambers, was demonstrated by the vehicle-treated mice and was considerably attenuated for bioal-

82 ERIKSSON AND FREDRIKSSON

TABLE I

EFFECTS OF NEONATAL EXPOSURE TO BIOALLETHRIN AND DELTAMETHRIN ON THE DENSITIES OF MUSCARINIC

RECEFTORS IN THE CEREBRAL CORTEX. HIPPOCAMPUS, AND STRIATUM OF THE ADULT MOUSE”

Cortex Hippocampus Striatum

Control 1266 -c 82 1332 f 134 1539 + 91 (12) (14) (10)

Bioallethrin 1132 f 1886 1377 * 108 1477 f 127 (15) (15) (9)

Deltamethrin 1207 * 88’ 1354+ 76 1546 rt_ 122 (18) (13) (13)

’ Ten-day-old male NMRI mice received either bioauethrin (0.7 mg), deltamethrin (0.7 mg), or the vehicle (10 ml) per kilogram of body weight orally once daily for 7 days. The mice were killed at an adult age of about 4 months. [3H]QNB binding (pmol/g protein, mean + SD) was assessed in the P2 fraction according to Nordberg and Winblad (198 1). The statistical evaluation was made by Student’s t test.

b p Q 0.05, compared with control. ’ 0.05 G p g 0.1, compared with control.

lethrin-treated mice. A hyperactive condition was also observed in deltamethrin-treated mice. In contrast to the observations made in the adult mouse, the behavioral test, loco- motion, in the neonatal mouse, 24 hr after the last administration, shows no significant changes between the bioallethrin-, deltameth- rin-, and vehicle-treated mice. Recently we re- ported that neonatal exposure to bioallethrin and deltamethrin, administered in the same manner and at same doses as in the present study, affected the MAChR in the 17-day-old

mouse (Eriksson and Nordberg, 1990). The bioallethrin exposure caused an increase in the density of MAChR in the cerebral cortex and an increase and decrease in the proportions of LA- and HA- binding sites, respectively. Del- tamethrin treatment also caused an increase in the density of MAChR, but the proportions of HA- and LA-binding sites were reversed. It is tempting to suggest that the differing changes in receptors between neonatal and adult mice following neonatal exposure to xenobiotics, that are not reflected in behavior, are an

TABLE 2

EFFECTS OF NEONATAL EXPOSURE TO BIOALLETHRIN AND DELTAMETHRIN ON HIGH- AND Low AFFINITY MUSCARINIC BINDING SITES (%) AND AFFINITY CONSTANTS (k) IN THE CEREBRAL CORTEX OF THE ADULT MOUSE”

High-affinity site Low-affinity site

Treatment n % k (PM) % k (FM)

Control 11 19.8 + 8.5 2.9 80.2 + 8.5 270 Bioallethrin 13 26.7 t- 14.3 3.0 73.2 rt 14.3 303 Deltamethrin 8 17.7 f 6.3 2.0 82.3 L 6.3 235

’ Ten-day-old male NMRI mice received either bioallethrin (0.7 mg), deltamethrin (0.7 mg). or the vehicle (10 ml) per kilogram of body weight once daily for 7 days. The mice were killed at an adult age of about 4 months. The binding parameters were estimated from [‘H]QNB/carbachol competition curves performed on the P2 fractions (see Methods). The percentage values are means f SD and the affinity constants are geometric means. The statistical evaluation between the pyrethroid-treated mice and controls of the percentage values and the affinity constants were made with Student’s t test and the Mann-Whitney U test, respectively. All p values exceeded 0.1.

PYRETHROIDS: NEONATAL AND ADULT NEUROTOXICITY 83

expression of receptor plasticity in the neonatal mouse and hence of its ability to cope with challenges of an environmental nature. With advancing age, this alteration in homeostatic compensatory mechanisms seems to turn into a state where the change in subpopulations of MAChR is reduced.

The consequence of this neonatal exposure to pyrethroids seems quite different from that reported in rodents exposed to pyrethroids as adults. Several reports have shown a decrease in locomotor activity and operant or schedule responding in adult rodents exposed to Type I or II pyrethroids in acute experiments (Bloom et al., 1983; Crofton and Reiter, 1984, 1987, 1988; Mitchell et al., 1988; Glowa, 1986; Peele and Crofton, 1987; Stein et al., 1987). These studies showed a dose-dependent de- crease in these behavioral variables. Crofton and Reiter (1984) have also reported that no significant cumulative effects could be ob- served on motor activity in the adult rat after a 30-day exposure to 2 mg/kg bw/day of del- tamethrin (Type II) or 6 mg/kg bw/day cis- methrin (Type I). In contrast to these reports the present result shows similarities to our earlier observation regarding the consequence of neonatal exposure to DDT. The changes in MAChR in neonatal mouse brain following DDT exposure were in agreement with those observed after bioallethrin treatment, showing an increase in the density of MAChR and in the proportion of LA-binding sites (Eriksson and Nordberg, 1986, 1990). At the adult age of 4-5 months, the cholinergic system was still affected, showing an increase in ACh release, a tendency toward a decrease in the density of MAChR, and the DDT-treated mice also dis- played a hyperactive condition (Eriksson et al., 1990a,b). From neurophysiological experi- ments in vitro it has been concluded that one of the basic mechanisms of neurotoxic action that pyrethroids and DDT have in common is their interference with the nerve membrane sodium channels (Lund and Narahashi, 1983; Van den Bercken and Vijverberg, 1980; Vijverberg and van den Bercken, 1982; Vijverberg et al., 1982; Narahashi, 1982,

1985). Type I pyrethroids and DDT induce large depolarizing afterpotentials, generating repetitive firing of the nerve, while Type II py- rethroids can produce a membrane depolar- ization, leading to a conduction block. These effects will disturb the synaptic transmission. Disturbance of action potentials during the period of synaptogenesis might be critical for normal brain development (Kalil, 1989). The change in cholinergic receptors and behavior in the adult mouse in the present study might therefore be the consequence of an early in- terference with the synaptic transmission in cholinergic nerve cells. However, it cannot be excluded that an interaction of the insecticides with the cholinergic receptors may occur dur- ing their rapid development in the neonatal mouse.

In conclusion, the present study provides further support for the hypothesis that during perinatal brain growth there is another critical period, comparable to the period of organo- genesis, when xenobiotics can produce tera- togenic effects, since bioallethrin, deltameth- rin, DDT, and DDOH-PA have all been found to affect the choline& system (Eriksson and Nordberg, 1986, 1990) during its rapid devel- opment, leading to permanent changes in the animals as adults, as observed earlier (Eriksson et al., 1990a,b) and in the present study.

ACKNOWLEDGMENTS

The authors thank Dr. Tessier, Rousell Uclaf, France, for the gift of deltamethrin and bioallethrin and Miss Anna Pettersson for excellent technical assistance. This work was financially supported by grants from the Swedish Envi- ronmental Protection Board and the Bank of Sweden Ter- centenary Foundation.

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